Mechanisms of beta-lactam antibiotics resistance will be investigated. New methodologies in the area combining quantum mechanics and molecular mechanics calculations, free energy perturbations, and continuum electrostatics models will be applied to examine the hydrolytic activity against beta-lactam antibiotics displayed by the bacterial defensive enzymes which render bacterial resistance-TEM-1 from E. coli and E. cloacae P99 beta-lactamases, which are common in clinical pathogens. The mechanism of catalysis will be first studied on simple models of methanol-mediated hydrolysis of small beta-lactam molecules in the gas phase and in the solution followed by the modeling for the methanol- mediated hydrolysis of benzylpenicillin-a typical beta-lactam antibiotic. Titration curves will be calculated for the representative beta-lactamases in order to elucidate mechanisms of activation of the critical active-site residues in these enzymes. Ultimately, the entire mechanism of the beta-lactam antibiotic hydrolysis by beta-lactamases will be studied. The detailed knowledge gained from the science disclosed in this proposal should ultimately be a driving force in designing novel potent antimicrobials that overcome the problem of bacterial resistance.